Method of detecting and identifying circulating antigens in human biological samples

ABSTRACT

Disclosed herein is a method of detecting and identifying antigens that are shed into human bodily fluids during infection. The disclosed method allows circulating antigens associated with a particular infection to be detected within minutes or hours from testing as compared to days required with the current methods. Methods of identifying diagnostic indicators/targets for a given condition or disease are disclosed which include immunizing a verterinary subject with biological fluids obtained from a human infected with particular antigens to identify diagnostic targets for immunoassay. Also disclosed are methods of diagnosing and monitoring a  B. pseudomallei -associated condition, such as melioidosis. Point-of-care immunoassays are also provided that can be used to diagnose or monitor the efficacy of a  B. pseudomallei -associated condition treatment. These immunoassays can also be used for rapid diagnosis of infection produced by  B. pseudomallei , such as meliodosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/804,621, filed Mar. 14, 2013, which claims the benefit of priorityunder 35 U.S.C. §119 to U.S. Provisional Application No. 61/675,977,filed on Jul. 26, 2012, both of which are incorporated herein byreference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. AI065359awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD

This relates to the field of antigen detection and specifically todetecting and identifying antigens circulating in human biologicalsamples.

BACKGROUND

Early diagnosis is critical for treatment of an infection to beeffective. Diagnostic assays that are capable of detecting low levels ofa particular molecule, such as an antigen, could greatly impact patientoutcome because they would be able to detect the molecule and thus acondition associated with such within minutes or hours from testing ascompared to days required with the current methods. Earlier detectiontranslates into earlier administration of therapies which couldsignificantly increase the likelihood of patient survival as well asdecrease the severity of the disease.

Current diagnostic tools are limited and diagnosis with these methodsoften occurs when the infection is so severe that treatment isinefficient and ineffective. For example, diagnosing infections, such asbacterial and fungal infections, is often plagued by symptoms of theparticular infection being non-specific making it difficult to obtain anaccurate diagnosis at the onset of the disease. Current diagnosticassays often can only detect a particular molecule, such as an antigen,associated with a particular disease or condition if such molecule ispresent at high levels, thus only detecting the infection associatedwith the particular molecule not until the infection is well developed.

SUMMARY

Disclosed herein is a method of detecting and identifying antigens thatare shed into human bodily fluids during infection. The disclosed methodallows circulating antigens associated with a particular infection to bedetected within minutes or hours from testing as compared to daysrequired with the current methods. Earlier detection translates intoearlier administration of therapies which increases the likelihood ofpatient survival as well as decreases the severity of the disease.

The disclosed method utilizes In vivo Microbial Antigen Discovery(InMAD). In contrast to studies previously utilizing InMAD whichemployed serum or urine from laboratory animals infected with aparticular antigen to immunize a laboratory animal for identifycirculating or secreted microbial antigens, the present method immunizesa laboratory animal with biological fluids obtained from a humaninfected with particular antigens to identify a diagnostic target forimmunoassay. The inventors made the suprising discovery that biologicalsamples collected from infected humans could be used to immunize alaboratory animal to identify circulating or secreted microbialantigens. One distinct advantage to the present method is that itcontrols for the possibility that the antigens associated with aparticular infection in a verterinary subject model may not alwayscorrelate with that of a human. The human biological samples containprecisely the antigens that are targets for immunoassay for a giveninfection. Identification of the targets allows a diagnostic assay to bedeveloped for a given condition in a human. It also allows for theeffectiveness of a treatment for a given subject and/or group ofsubjects for a particular infection to be monitored.

In one embodiment of the InMAD technique, biological fluid samples, suchas serum and urine samples, are collected from subjects afflicted with agiven infection. Biological fluid samples are filtered to remove wholecells, but leave behind soluble antigens released during infection. Thefiltered samples are used to immunize naïve verterinary subjects andtheir serum is collected to identify antigens recognized by antibodiesusing one dimensional and two dimensional immunoblots prepared fromwhole cell lysates. Mass spectroscopy is used to identify those reactiveantigens.

Based on these findings, disclosed herein are methods of identifyingdiagnostic indicators. In some embodiments, these methods includeselecting a condition or disease for which a diagnostic assay is desiredand is believed to be associated with one or more antigens; immunizing averterinary subject which is not afflicted with the selected conditionor disease with a human biological sample obtained from a human subjecthaving the selected condition or disease; detecting one or more antigensin a biological sample obtained from the immunized animal subject;comparing the one or more antigens detected in the immunized animalsubject sample with a control; and identifying one or more diagnosticindicators for the selected condition or disease, wherein an alterationin at least one antigen detected in the sample obtained from theimmunized subject relative to the control indicates that such antigen isa diagnostic indicator for the condition or disease.

In some embodiments, the method further includes obtaining thebiological sample, such as serum or urine, from the human subject withthe selected condition or disease.

In some embodiments, the method further includes filtering the humanbiological sample obtained from the human subject to isolate the one ormore soluble antigens.

In some embodiments, the method further includes obtaining thebiological sample, such as serum or urine, from the immunized animalsubject prior to detecting one or more antigens.

In some embodiments of the method, detecting one or more antigens in abiological sample obtained from the immunized animal subject includesusing one-dimensional or two-dimensional immunoblots followed by massspectroscopy to identify the one or more antigens.

Also disclosed are methods of diagnosing and monitoring anantigen-associated condition, such as Burkholderiapseudomallei-associated condition including melioidosis. In one example,the disclosed methods allow for self monitoring in which a subject, suchas an immunosuppressed patient, monitors the presence of one or morespecific antigens, to monitor the onset of an infection.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of an exemplary InMAD strategy foridentification of targets for immunoassay by using human biologicalsamples.

FIG. 2A is a digital image of a two-dimensional blot of Burkholderiapseudomallei lysate probed with InMAD immune serum illustrating thetotal proteins from the Burkholderia pseudomallei.

FIG. 2B is digital image of a two-dimensional Western blot of B.pseudomallei lysate probed with InMAD immune serum from mice immunizedwith human urine. Twelve spots (proteins) not found after immunizationwith control serum or urine from uninfected animals are circled. Eachspot is a candidate diagnostic target. Each number corresponds to theprotein number in Table 1 all of which were identified by massspectroscopy.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

I. Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology may be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. The term“comprises” means “includes.” All GenBank and Protein ID Nos.,publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Alteration or difference: An increase or decrease in the amount of something, such as a protein antigen. In some examples, the difference isrelative to a control or reference value or range of values, such as anamount of a protein that is expected in a subject who does not have aparticular condition or disease being evaluated. Detecting an alterationor differential expression/activity can include measuring a change inprotein expression, concentration or activity, such as by ELISA, Westernblot and/or mass spectrometry.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, mice.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically binds anepitope of a protein listed in the tables below, or a fragment of any ofthese proteins. Antibodies can include a heavy chain and a light chain,each of which has a variable region, termed the variable heavy (VH)region and the variable light (VL) region. Together, the VH region andthe VL region are responsible for binding the antigen recognized by theantibody. This includes intact immunoglobulins and the variants andportions of them well known in the art, such as Fab′ fragments, F(ab)′2fragments, single chain Fv proteins (“scFv”), and disulfide stabilizedFv proteins (“dsFv”). A scFv protein is a fusion protein in which alight chain variable region of an immunoglobulin and a heavy chainvariable region of an immunoglobulin are bound by a linker, while indsFvs, the chains have been mutated to introduce a disulfide bond tostabilize the association of the chains. The term also includesrecombinant forms such as chimeric or humanized antibodies that may bederived from a murine antibody, heteroconjugate antibodies (such as,bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.); Kuby, Immunology, 3rd Ed., W.H.Freeman & Co., New York, 1997.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. These fused cells and their progeny are termed“hybridomas.” Monoclonal antibodies include humanized monoclonalantibodies.

A variety of immunoassay formats are appropriate for selectingantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow & Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York (1988), for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. An “antigenic polypeptide” is apolypeptide to which an immune response, such as a T cell response or anantibody response, can be stimulated. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and multi-dimensionalnuclear magnetic resonance spectroscopy. The term “antigen” denotes bothsubunit antigens, (for example, antigens which are separate and discretefrom a whole organism with which the antigen is associated in nature),as well as killed, attenuated or inactivated bacteria, viruses, fungi,parasites or other microbes. An “antigen,” when referring to a protein,includes a protein with modifications, such as deletions, additions andsubstitutions (generally conservative in nature) to the native sequence,so long as the protein maintains the ability to elicit an immunologicalresponse, as defined herein. These modifications may be deliberate, asthrough site-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the antigens.

Bacteria: A large domain of prokaryotic microorganisms. Typically a fewmicrometres in length, bacteria have a wide range of shapes, rangingfrom spheres to rods and spirals. There are broadly speaking twodifferent types of cell wall in bacteria, called Gram-positive andGram-negative. Gram-positive bacteria possess a thick cell wallcontaining many layers of peptidoglycan and teichoic acids. In contrast,Gram-negative bacteria have a relatively thin cell wall consisting of afew layers of peptidoglycan surrounded by a second lipid membranecontaining lipopolysaccharides and lipoproteins. Most bacteria have theGram-negative cell wall, and only the Firmicutes and Actinobacteria havethe alternative Gram-positive arrangement. An example of a Gram-negativebacterium is Burkholderia pseudomallei. A “Burkholderiapseudomallei-associated molecule” is a molecule associated with one ormore signs or symptoms of melioidosis. In some examples, amelioidosis-associated molecule is one or more of the antigens providedin Table 1.

Contacting: “Contacting” includes in solution and solid phase, forexample contacting a salivary protein with a test agent. The test agentmay also be a combinatorial library for screening a plurality ofcompounds. In another example, contacting includes contacting a samplewith an antibody, for example contacting a sample that contains aprotein of interest such as a protein associated with a particularcondition or disease.

Diagnosis: The process of identifying a condition or disease by itssigns, symptoms, results of various tests and presence of diagnosticindicators. The conclusion reached through that process is also called“a diagnosis.”

Electron Transfer flavoprotein subunit alpha (ETFA): The alpha subunitof the electron transfer flavoprotein (ETF). ETFs are heterodimericproteins composed of an alpha and beta subunit (ETFA and ETFB), andcontain an FAD cofactor and AMP. ETF has three domains: domains I and IIare formed by the N- and C-terminal portions of the alpha subunit,respectively, while domain III is formed by the beta subunit.

ETFA is encoded by the ETFA gene. ETFA participates in catalyzing theinitial step of the mitochondrial fatty acid beta oxidation. It shuttleselectrons between primary flavoprotein dehydrogenases and themembrane-bound electron transfer flavoprotein ubiquinone oxidoreductase.Defects in electron-transfer-flavoprotein have been implicated in typeII glutaricaciduria in which multiple acyl CoA dehydrogenasedeficiencies result in large excretion of glutaric, lactic,ethylmalonic, butyric, isobutyric, 2-methyl-butyric, and isovalericacids. ETFA is detected in a subject with B. pseudomallei and can beused to diagnosis or monitor such condition.

Exemplary nucleic acid and protein sequences for ETFA are publiclyavailable (see, Entrez No. 2108 or GenBank No. NM_(—)000126.3 for humanETFA nucleic acid sequences; Entrez No. 110842 or GenBank No.NM_(—)145615.4 for mouse ETFA nucleic acid sequences; and GenBank Nos.NP_(—)000117.1 (human) and NP_(—)663590.3 (mouse) for ETFA proteinsequences; each of which hereby incorporated by reference as availableon Jul. 26, 2012).

In one example, ETFA includes a full-length wild-type (or native)sequence, as well as ETFA allelic variants, fragments, homologs orfusion sequences that retain the ability to be detected in a subjectwith melioidosis. In certain examples, ETFA has at least 80% sequenceidentity, for example at least 85%, 90%, 95%, or 98% sequence identityto a known ETFA and retains ETFA activity (e.g., the capability to bedetected in a subject with melioidosis). ETFA is detected in a subjectwith B. pseudomallei and can be used to diagnosis or monitor suchcondition.

FtsA/FtsZ: FtsA is a protein encoded by the FtsA gene and participatesin formation of the Escherichia coli septum structure. FtsZ is a proteinencoded by the ftsZ gene that assembles into a ring at the future siteof the septum of bacterial cell division. FtsA and FtsZ are involved incell divisiona. FtsA/FtsZ has been named after “Filamentingtemperature-sensitive mutant A” whereas FtsZ is for “Filamentingtemperature-sensitive mutant Z.”

Exemplary nucleic acid and protein sequences for FtsA/FTsZ are publiclyavailable (see, GenBank Nos. gi 53720630/53720631, each of which herebyincorporated by reference as available on Jul. 26, 2012).

In one example, FtsA/FtsZ includes a full-length wild-type (or native)sequence, as well as FtsA/FtsZ allelic variants, fragments, homologs orfusion sequences that retain the ability to be detected in a subjectwith melioidosis. In certain examples, FtsA/FtsZ has at least 80%sequence identity, for example at least 85%, 90%, 95%, or 98% sequenceidentity to a known FtsA/FtsZ and retains FtsA/FtsZ activity (e.g., thecapability to be detected in a subject with melioidosis). FtsA/FtsZ aredetected in a subject with B. pseudomallei and can be used to diagnosisor monitor such condition.

Fungus: Living, single-celled and multicellular organisms belonging tothe kingdom Fungi. Most species are characterized by a lack ofchlorophyll and presence of chitinous cell walls, and some fungi may bemultinucleated. The methods disclosed herein can be used to detect andidentify antigens associated with particular fungi. In one example, themethod is used to detect a fungus, such as an Aspergillus species.Representative, non-limiting examples of Aspergillus species include A.candidus, A. chevalieri, A. clavatus, A. flavipes, A. flavus, A.fumigatus, A. granulosus, A. nidulans, A. niger, A. parasiticus, A.restrictus, A. sydowii, A. tamari, A. ustus, A. versicolor, and A.wentii.

GroEL: A protein that belongs to the alkalai family of molecularchaperones, and is found in a large number of bacteria. It is known toplay a role in protein folding. To function properly, GroEL requires thelid-like co-chaperonin protein complex GroES. In eukaryotes, the proteinHsp60 is believed to be structurally and functionally nearly identicalto GroEL.

Exemplary nucleic acid and protein sequences for GroEL are publiclyavailable (see, GenBank Nos. NM_(—)002156 (human) and NM_(—)010477(mouse) for GroEL nucleic acid sequences and NP_(—)002147 (human) andNP_(—)034607 (mouse) for GroEL protein sequences; each of which herebyincorporated by reference as available on Jul. 26, 2012).

In one example, GroEL includes a full-length wild-type (or native)sequence, as well as GroEL allelic variants, fragments, homologs orfusion sequences that retain the ability to be detected in a subjectwith melioidosis. In certain examples, GroEL has at least 80% sequenceidentity, for example at least 85%, 90%, 95%, or 98% sequence identityto a known GroEL and retains GroEL activity (e.g., the capability to bedetected in a subject with melioidosis).

Glutathione S-transferase (GST) domain containing protein: A structuraldomain of glutathione S-transferase (GST). GST conjugates reducedglutathione to a variety of targets including S-crystallin from squid,the eukaryotic elongation factor 1-gamma, the HSP26 family ofstress-related proteins and auxin-regulated proteins in plants. Theglutathione molecule binds in a cleft between N and C-terminal domains.The catalytic residues are proposed to reside in the N-terminal domain.In eukaryotes, glutathione S-transferases (GSTs) participate in thedetoxification of reactive electrophilic compounds by catalysing theirconjugation to glutathione. The GST domain is also found inS-crystallins from squid and proteins with no known GST activity, suchas eukaryotic elongation factors 1-gamma and the HSP26 family ofstress-related proteins, which include auxin-regulated proteins inplants and stringent starvation proteins in Escherichia coli. BacterialGSTs of known function often have a specific, growth-supporting role inbiodegradative metabolism: epoxide ring opening andtetrachlorohydroquinone reductive dehalogenation are two examples of thereactions catalysed by these bacterial GSTs. Some regulatory proteins,like the stringent starvation proteins, also belong to the GST family.Glutathione S-transferases form homodimers, but in eukaryotes can alsoform heterodimers of the A1 and A2 or YC1 and YC2 subunits. Thehomodimeric enzymes display a conserved structural fold. Each monomer iscomposed of a distinct N-terminal sub-domain, which adopts thethioredoxin fold, and a C-terminal all-helical sub-domain. This entry isthe C-terminal domain.

Exemplary protein sequences for glutathione S-transferase domaincontaining protein are publicly available (see, GenBank No. gi 76808775,which hereby incorporated by reference as available on Jul. 26, 2012).

In one example, GST domain containing protein includes a full-lengthwild-type (or native) sequence, as well as GST domain containing proteinallelic variants, fragments, homologs or fusion sequences that retainthe ability to be detected in a subject with melioidosis. In certainexamples, GST domain containing protein has at least 80% sequenceidentity, for example at least 85%, 90%, 95%, or 98% sequence identityto a known GST domain containing protein and retains GST domaincontaining protein activity (e.g., the capability to be detected in asubject with melioidosis). GST domain containing protein is detected ina subject with B. pseudomallei and can be used to diagnosis or monitorsuch condition.

Immunoassay: A biochemical test that measures the presence orconcentration of a substance in a sample, such as a biological sample,using the reaction of an antibody to its cognate antigen, for examplethe specific binding of an antibody to a protein. Both the presence ofantigen and the amount of antigen present can be measured. For measuringproteins, for each the antigen and the presence and amount (abundance)of the protein can be determined or measured. Measuring the quantity ofantigen can be achieved by a variety of methods. One of the most commonis to label either the antigen or antibody with a detectable label.

An “enzyme linked immunosorbent assay (ELISA)” is type of immunoassayused to test for antigens (for example, proteins present in a sample,such as a biological sample). A “competitive radioimmunoas say (RIA)” isanother type of immunoassay used to test for antigens. A “lateral flowimmunochromatographic (LFI)” assay is another type of immunoassay usedto test for antigens.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages (such ashorseradish peroxidase), radioactive isotopes (for example ¹⁴C, ³²P,¹²⁵I, ³H isotopes and the like) and particles such as colloidal gold. Insome examples a protein, such as a protein associated with a particularinfection, is labeled with a radioactive isotope, such as ¹⁴C, ³²P,¹²⁵I, ³H isotope. In some examples an antibody that specifically bindsthe protein is labeled. Methods for labeling and guidance in the choiceof labels appropriate for various purposes are discussed for example inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 1989) and Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998), Harlow & Lane(Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, 1988).

Melioidosis: An infectious disease caused by a Gram-negative bacterium,Burkholderia pseudomallei, found in soil and water. It is of publichealth importance in endemic areas, particularly in Thailand andnorthern Australia. It exists in acute and chronic forms. Symptoms mayinclude pain in chest, bones, or joints; cough; skin infections, lungnodules and pneumonia. B. pseudomallei was thought to be a member of thePseudomonas genus and was previously known as Pseudomonas pseudomallei.It is phylogenetically related closely to Burkholderia mallei whichcauses glanders, an infection primarily of horses, donkeys and mules.

Acute melioidosis normally has an incubation period of less than amonth. Patients with latent melioidosis may be symptom free for decades.Chronic melioidosis is usually defined by a duration of symptoms greaterthan 2 months and occurs in approximately 10% of patients. The clinicalpresentation of chronic melioidosis is protean and includes suchpresentations as chronic skin infection, skin ulcers and lung nodules orchronic pneumonia. A patient with active melioidosis may present a feveror other pain or other symptoms such as cough, pleuritic chest pain,bone or joint pain, or intra-abdominal infection (including liver and/orsplenic abscesses, or prostatic abscesses)

A current treatment of melioidosis may be divided into two stages, anintravenous high intensity stage and an oral maintenance stage toprevent recurrence. The intravenous intensive phase may includeintravenous ceftazidime for treatment of acute melioidosis. Meropenem,imipenem and cefoperazone-sulbactam (Sulperazone) are also active.Intravenous amoxicillin-clavulanate (co-amoxiclav) may also be used.Intravenous antibiotics are typically given for a minimum of 10 to 14days, and are continued until the patient's temperature has returned tonormal for more than 48 hours: it is not uncommon for patients torequire parenteral treatment continuously for more than a month.Additional possible therapeutic agents include cefepime, ertapenem,piperacillin-sulbactam, doripenem and biapenem.

Following the treatment of the acute disease, a maintenance treatmentmay be provided to the patient such as administration of co-trimoxazoleand doxycycline for a period of time (such as 12 to 20 weeks) to reducethe rate of recurrence. Other maintenance treatments may includeadministration of chloramphenicol and co-amoxiclay.

The methods, compositions and assays disclosed herein provide a means ofidentifying a subject who has melioidosis or who is at increased risk ofdeveloping melioidosis. A “non-melioidosis” or “normal” subject does nothave any form of melioidosis.

A “melioidosis-associated molecule” is a molecule associated with one ormore signs or symptoms of melioidosis. In some examples, amelioidosis-associated molecule is one or more of the antigens providedin Table 1.

Microorganism: A single-celled, or unicellular, organism which includebacteria, fungi, archaea or protists, but not viruses and prions (whichare generally classified as non-living). Microorganisms that causedisease in a host are known as pathogens.

Transcription Elongation Factor NusA: A protein associated with B.pseudomallei and can be used to diagnosis or monitor a B. pseudomalleiinfection such as melioidosis. Exemplary nucleic acid and proteinsequences for transcription elongation factor NusA are publiclyavailable (see, for example, GenBank Nos. EIM96869.1, AEW69130.1,YP_(—)542577.1, and ZP_(—)10253383.1 for exemplary protein sequences,each of which hereby incorporated by reference as available on Jul. 26,2012).

In one example, transcription elongation factor NusA includes afull-length wild-type (or native) sequence, as well as transcriptionelongation factor NusA allelic variants, fragments, homologs or fusionsequences that retain the ability to be detected in a subject withmelioidosis. In certain examples, transcription elongation factor NusAhas at least 80% sequence identity, for example at least 85%, 90%, 95%,or 98% sequence identity to a known transcription elongation factor NusAand retains transcription elongation factor NusA activity (e.g., thecapability to be detected in a subject with melioidosis). Transcriptionelongation factor NusA is detected in a subject with B. pseudomallei andcan be used to diagnosis or monitor such condition.

II. Methods for Detecting and Identifying Circulating Antigens

Disclosed herein are methods for detecting and identifying circulatingantigens that can be used to identify diagnostic indicators/targets ofspecific conditions and/or diseases. In one example, a method ofidentifying one or more diagnostic indicators includes selecting acondition or disease for which a diagnostic assay is desired and isbelieved to be associated with one or more antigens. For example, thecondition can be one that is associated with a particular set ofclinical factors/symptoms or presence of a microorganism such a fungusor bacteria.

The method for identifying one or more diagnostic indicators alsoincludes immunizing a verterinary subject (such as a mouse or rabbit)that is not afflicted with the selected condition or disease with ahuman biological sample obtained from a human subject having theselected condition or disease. For example, a biological sample, such asurine, is collected from a human subject displaying one or more signs orsymptoms associated with the selected condition or disease for which adiagnostic assay is desired. In other examples, other biological fluids,such as blood (such as whole blood obtained from a finger prick), GCF,amniotic fluid, BALF, salvia or tears are collected. In someembodiments, the method further includes filtering the human biologicalsample obtained from the human subject to isolate the one or moresoluble antigens present in the sample.

The disclosed method for identifying one or more diagnosticindicators/targets also includes detecting one or more antigens in abiological sample obtained from the immunized animal subject; comparingthe one or more antigens detected in the immunized animal subject samplewith a control; and identifying one or more diagnostic indicators forthe selected condition or disease, wherein an alteration in at least oneantigen detected in the sample obtained from the immunized subjectrelative to the control indicates that such antigen is a diagnosticindicator for the condition or disease. In some examples, the methodfurther includes obtaining the biological sample, such as serum orurine, from the immunized animal subject prior to detecting one or moreantigens. In some embodiments of the method, detecting one or moreantigens in a biological sample obtained from the immunized animalsubject includes using one-dimensional or two-dimensional immunoblotsfollowed by mass spectroscopy to identify the one or more antigens.

In some examples, the method includes detecting an increase, such as astatistically significant increase, such as an at least a 1.5, 2, 3, 4,or 5 fold increase in the amount of one or more molecules associatedwith condition or disease, including at least a 1.5, 2, 3, 4, or 5 foldincrease to a control or reference value, such as between a 1.5 to 5fold increase, a 2 to 6 fold increase, a 3 to 10 fold increase,including a 2 fold, a 3 fold, a 4 fold, a 5 fold, a 6 fold, a 7 fold, a8 fold, a 9 fold or 10 fold increase. In some embodiments, the methodincludes detecting a decrease, such as a statistically significantdecrease, such as at least a 2, 3, 4, or 5 fold decrease in the amountof one or more molecules associated with the selected condition ordisease, such as one or more protein antigens, as compared to a controlor reference sample, such as between a 1.5 to 5 fold decrease, a 2 to 6fold decrease, a 3 to 10 fold decrease, including a 2 fold, a 3 fold, a4 fold, a 5 fold, a 6 fold, a 7 fold, a 8 fold, a 9 fold or 10 folddecrease.

In some embodiments of the method, the disclosed methods allow for selfmonitoring in which a subject, such as an immuno suppressed patient,monitors the presence of one or more specific antigens, to monitor theonset of an infection.

III. Methods for Detecting B. pseudomallei-Associated Condition andMonitoring the Efficacy of a Therapeutic Regimen

Methods are disclosed herein that are of use to determine if a subjecthas a B. pseudomallei-associated condition, such as melioidosis,including acute or chronic melioidosis, or to monitor the efficacy oftherapy. These methods utilize a biological fluid, such as, but notlimited to urine or serum, for the detection of a molecule associated B.pseudomallei, such as melioidosis, including, but not limited to,protein antigens listed in Table 1. The B. pseudomallei-associatedmolecules, such as melioidosis-associated molecules, include anynaturally occurring forms of the proteins, such as but not limited toglycosylated forms. In some embodiments, the methods disclosed hereinare used to identify a subject as having acute melioidosis. In otherembodiments, the methods are used to identify chronic melioidosis. Ineven further embodiments, the methods are used to diagnosis latentmelioidosis. These methods can be performed over time, to monitor theprogression or regression of melioidosis in a subject, or to assess forthe development of melioidosis from a pre-melioidosis condition. In someexamples, the disclosed methods are used for self monitoring in which asubject, such as an immunosuppressed human subject, monitors thepresence of one or more molecules associated with B. pseudomallei, suchas melioidosis, to monitor the onset of an infection. In additionalexamples, the disclosed methods and kits are used for self monitoring inwhich a subject, such as a subject that has previously been diagnosedand treated for a melioidosis associated condition or disease practicesthe method or uses the kit to monitor for relapse.

Methods are disclosed herein that include testing a biological sample,such as a serum or urine sample, obtained from a human at risk orsuspected of having melioidosis. In one example, the biological sampleis a biological fluid, such as urine. However, other biological fluidsare also of use, such as blood (such as whole blood obtained from afinger prick), GCF, amniotic fluid, BALF, salvia or tears. The methodsinclude detecting, or determining the abundance (amount) of one or moremolecules associated with melioidosis, including protein antigens listedin Table 1. In some examples, the methods include determining aproteomic profile.

In one example, the method includes detecting at least GroEL. In oneexample, the method includes detecting GroEL and at least one moremolecule associated with melioidosis such as one or more moleculeslisted in Table 1. The methods can include detecting at least one, suchas at least two, at least three, at least four, at least five, at leastsix, at least seven, at least eight, at least nine, at least ten, atleast eleven, at least twelve, including one, two, three, four, five,six, seven, eight, nine, ten, eleven, or twelve molecules associatedwith melioidosis. In one example, the method includes detecting at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, including one, two, three, four, five,six, seven, eight, nine, ten, eleven, or twelve molecules listed inTable 1.

In some embodiments, the method includes detecting an increase, such asa statistically significant increase, such as an at least a 1.5, 2, 3,4, or 5 fold increase in the amount of one or more molecules associatedwith melioidosis, including at least a 1.5, 2, 3, 4, or 5 fold increasein one or more protein antigens listed in Table 1 as compared to areference value. In some embodiments, the method includes detecting adecrease, such as a statistically significant decrease, such as at leasta 2, 3, 4, or 5 fold decrease in the amount of one or more proteinantigens listed in Table 1 as compared to a reference sample.

In one embodiment, the method includes comparing a proteomic profile ofa test sample of urine from a human subject of interest comprising atleast one of protein associated with melioidosis, such a protein antigenlisted in Table 1, with a proteomic profile from a reference sample.

In one embodiment, the method determines if the human subject hasmelioidosis. If the reference sample is a normal sample and theproteomic profile of the test sample is essentially the same as theproteomic profile of the normal sample, the human subject is determinednot to have melioidosis. However, if the proteomic profile of the testsample has a unique expression signature relative to the proteomicprofile of the normal sample the human subject is determined to havemelioidosis.

In some embodiments, if the reference sample is a sample from a humansubject with melioidosis, and its proteomic profile shares at least oneunique expression signature characteristic with the reference sample,then the human subject is determined to have melioidosis. If theproteomic profile of the test sample has a unique expression signaturerelative to the reference sample the human subject is determined not tohave melioidosis. Hence, the proteomic profile provides an additionaldiagnostic criterion for these disorders.

In one embodiment, the method is a method to determine if a therapy iseffective for the treatment of the human subject by detecting thepresence of at least one protein associated with melioidosis. The methodcan be performed multiple times over a specified time period, such asdays, weeks, months or years. In several examples, the therapy includestreatment with a therapeutic agent for melioidosis. If the referencesample is a normal human sample, and the proteomic profile of the testsample is essentially the same as the proteomic profile of the normalsample the human subject is determined to have an effective therapy,while if the proteomic profile of the test sample has a uniqueexpression signature relative to the proteomic profile of the normalsample to have an ineffective therapy. If the reference sample is asample from a human subject with melioidosis, and proteomic profileshares at least one unique expression signature characteristic with thereference sample then the human subject is determined to have anineffective therapy, while if the proteomic profile of the test samplehas a unique expression signature relative to the reference sample thehuman subject is determined to have an effective therapy. Changes in theprofile can also represent the progression (or regression) of thedisease process. Methods for monitoring the efficacy of therapeuticagents are described below.

Monitoring

The diagnostic methods of the present disclosure are valuable tools forpracticing physicians to make quick treatment decisions for melioidosisconditions, including both acute and chronic melioidosis. Thesetreatment decisions can include the administration of ananti-melioidosis agent and decisions to monitor a subject for onsetand/or advancement of melioidosis. The method disclosed herein can alsobe used to monitor the effectiveness of a therapy.

Following the measurement of the expression levels of one or more of themolecules identified herein, the assay results, findings, diagnoses,predictions and/or treatment recommendations are typically recorded andcommunicated to technicians, physicians and/or patients, for example. Incertain embodiments, computers will be used to communicate suchinformation to interested parties, such as, patients and/or theattending physicians. Based on the measurement, the therapy administeredto a subject can be modified.

In one embodiment, a diagnosis, prediction and/or treatmentrecommendation based on the expression level in a test subject of one ormore of the melioidosis associated molecules disclosed herein iscommunicated to the subject as soon as possible after the assay iscompleted and the diagnosis and/or prediction is generated. The resultsand/or related information may be communicated to the subject by thesubject's treating physician. Alternatively, the results may becommunicated directly to a test subject by any means of communication,including writing, such as by providing a written report, electronicforms of communication, such as email, or telephone. Communication maybe facilitated by use of a computer, such as in case of emailcommunications. In certain embodiments, the communication containingresults of a diagnostic test and/or conclusions drawn from and/ortreatment recommendations based on the test, may be generated anddelivered automatically to the subject using a combination of computerhardware and software which will be familiar to artisans skilled intelecommunications. One example of a healthcare-oriented communicationssystem is described in U.S. Pat. No. 6,283,761; however, the presentdisclosure is not limited to methods which utilize this particularcommunications system. In certain embodiments of the methods of thedisclosure, all or some of the method steps, including the assaying ofsamples, diagnosing of diseases, and communicating of assay results ordiagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.

In several embodiments, identification of a subject as havingmelioidosis results in the physician treating the subject, such asprescribing one or more therapeutic agents for inhibiting or delayingone or more signs and symptoms associated with melioidosis. Inadditional embodiments, the dose or dosing regimen is modified based onthe information obtained using the methods disclosed herein.

The subject can be monitored while undergoing treatment using themethods described herein in order to assess the efficacy of thetreatment protocol. In this manner, the length of time or the amountgive to the subject can be modified based on the results obtained usingthe methods disclosed herein.

IV. Immunoassays for Diagnosing and Monitoring B.pseudomallei-associated Conditions

The methods disclosed herein can be performed in the form of variousimmunoassay formats, which are well known in the art. There are two maintypes of immunoassays, homogeneous and heterogeneous. In homogeneousimmunoassays, both the immunological reaction between an antigen and anantibody and the detection are carried out in a homogeneous reaction.Heterogeneous immunoassays include at least one separation step, whichallows the differentiation of reaction products from unreacted reagents.A variety of immunoassays can be used to detect one or more of themolecules capable of detecting a B. pseudomallei-associated molecule,including detecting extracellular polysaccharides. In one example, oneor more antigens associated with an B. pseudomallei-associateddisorder/condition are measured to diagnose an B.pseudomallei-associated disorder, such as melioidosis. For example, oneor more antigens listed in Table 1 are detected with a disclosedimmunoassay. In some examples, the disclosed immunoassay includes atleast one, such as two, three, four, five, six, seven, eight, nine, ten,eleven, or more molecules associated with a B. pseudomallei-associatedcondition or disease, such as melioidosis. In one example, theimmunoassay includes at least one, such as two, three, four, five, six,seven, eight, nine, ten, or eleven molecules listed in Table 1.

ELISA is a heterogeneous immunoassay, which has been widely used inlaboratory practice since the early 1970s, and can be used in themethods disclosed herein. The assay can be used to detect proteinantigens in various formats. In the “sandwich” format the antigen beingassayed is held between two different antibodies. In this method, asolid surface is first coated with a solid phase antibody. The testsample, containing the antigen (e.g., a diagnostic protein), or acomposition containing the antigen, such as a urine sample from asubject of interest, is then added and the antigen is allowed to reactwith the bound antibody. Any unbound antigen is washed away. A knownamount of enzyme-labeled antibody is then allowed to react with thebound antigen. Any excess unbound enzyme-linked antibody is washed awayafter the reaction. The substrate for the enzyme used in the assay isthen added and the reaction between the substrate and the enzymeproduces a color change. The amount of visual color change is a directmeasurement of specific enzyme-conjugated bound antibody, andconsequently the antigen present in the sample tested.

ELISA can also be used as a competitive assay. In the competitive assayformat, the test specimen containing the antigen to be determined ismixed with a precise amount of enzyme-labeled antigen and both competefor binding to an anti-antigen antibody attached to a solid surface.Excess free enzyme-labeled antigen is washed off before the substratefor the enzyme is added. The amount of color intensity resulting fromthe enzyme-substrate interaction is a measure of the amount of antigenin the sample tested. A heterogeneous immunoassay, such as an ELISA, canbe used to detect any molecules associated with a B. pseudomalleiantigen.

In another example, immuno-PCR can be used to detect any of themolecules associated with a B. pseudomallei condition such asmelioidosis. Immuno-PCR is a modification of the conventional ELISAformat in which the detecting antibody is labeled with a DNA label, andis applicable to the analysis of biological samples (see, e.g., U.S.Pat. No. 5,665,539 and U.S. Patent Application Publication No.2005/0239108; all herein incorporated by reference). The amplificationability of PCR provides large amounts of the DNA label which can bedetected by various methods, typically gel electrophoresis withconventional staining (e.g., Sano et al., Science, 258:120-122, 1992).This method can also include the direct conjugation of the DNA label tothe antibody and replacement of gel electrophoresis by using labeledprimers to generate a PCR product that can be assayed by ELISA or usingreal time quantitative PCR. In an example of the real-time PCR method,PCR is used to amplify DNA in a sample in the presence of anonextendable dual labeled fluorogenic hybridization probe. Onefluorescent dye serves as a reporter and its emission spectra isquenched by the second fluorescent dye. The method uses the 5′ nucleaseactivity of Taq polymerase to cleave a hybridization probe during theextension phase of PCR. The nuclease degradation of the hybridizationprobe releases the quenching of the reporter dye resulting in anincrease in peak emission from the reporter. The reactions are monitoredin real time.

Homogeneous immunoassays include, for example, the Enzyme MultipliedImmunoassay Technique (EMIT), which typically includes a biologicalsample comprising the biomarkers to be measured, enzyme-labeledmolecules of the biomarkers to be measured, specific antibody orantibodies binding the biomarkers to be measured, and a specific enzymechromogenic substrate. In a typical EMIT, excess of specific antibodiesis added to a biological sample. If the biological sample contains themolecules to be detected, such molecules bind to the antibodies. Ameasured amount of the corresponding enzyme-labeled molecules is thenadded to the mixture. Antibody binding sites not occupied by moleculesof the protein in the sample are occupied with molecules of the addedenzyme-labeled protein. As a result, enzyme activity is reduced becauseonly free enzyme-labeled protein can act on the substrate. The amount ofsubstrate converted from a colorless to a colored form determines theamount of free enzyme left in the mixture. A high concentration of theprotein to be detected in the sample causes higher absorbance readings.Less protein in the sample results in less enzyme activity andconsequently lower absorbance readings. Inactivation of the enzyme labelwhen the antigen-enzyme complex is antibody-bound makes the EMIT auseful system, enabling the test to be performed without a separation ofbound from unbound compounds as is necessary with other immunoassaymethods. A homogenous immunoassay, such as an EMIT, can be used todetect any of the molecules associated with a B. pseudomallei-associatedcondition or disease, such as B. pseudomallei protein antigens listed inTable 1.

Immunoassay kits are also disclosed herein. These kits include, inseparate containers (a) monoclonal antibodies having binding specificityfor the polypeptides used in the diagnosis of an B.pseudomallei-associated condition/disorder, such as melioidosis; and (b)and anti-antibody immunoglobulins. This immunoassay kit may be utilizedfor the practice of the various methods provided herein. The monoclonalantibodies and the anti-antibody immunoglobulins can be provided in anamount of about 0.001 mg to 100 grams, and more preferably about 0.01 mgto 1 gram. The anti-antibody immunoglobulin may also be a polyclonalimmunoglobulin, protein A or protein G or functional fragments thereof,which may be labeled prior to use by methods known in the art. Inseveral embodiments, the immunoassay kit includes one, two, three orfour or more antibodies that specifically bind to molecules associatedwith a B. pseudomallei-associated condition or disease, such as B.pseudomallei protein antigens listed in Table 1. The immunoassay kit canalso include one or more antibodies that specifically bind to one ormore of these molecules. Thus, the kits can be used to detect one ormore different molecules associated an B. pseudomallei-associatedcondition, such as melioidosis.

Immunoassays for polysaccharides and proteins differ in that a singleantibody is used for both the capture and indicator roles forpolysaccharides due to the presence of repeating epitopes. In contrast,two antibodies specific for distinct epitopes are required forimmunoassay of proteins. Exemplary samples include biological samplesobtained from subjects including, but not limited to, serum, blood andurine samples. In some examples, an exemplary sample includesbronchoalveolar lavage fluid.

In one particular example, a quantitative ELISA is constructed fordetection of at least one of the B. pseudomallei protein antigens listedin Table 1. These immunoassays utilize antibodies, such as mAbscommercially available. Since a polysaccharide is a polyvalent repeatingstructure, a single mAb may be used for both the capture and indicatorphases of an immunoassay. The only requirement is that the mAb have asufficient affinity. A mAb with an affinity of about 0.5 μM hassufficient affinity.

V. Capture Device Methods

The disclosed methods can be carried out using a sample capture device,such as a lateral flow device (for example a lateral flow test strip)that allows detection of one or more molecules, such as those describedherein.

Point-of-use analytical tests have been developed for the routineidentification or monitoring of health-related conditions (such aspregnancy, cancer, endocrine disorders, infectious diseases or drugabuse) using a variety of biological samples (such as urine, serum,plasma, blood, saliva). Some of the point-of-use assays are based onhighly specific interactions between specific binding pairs, such asantigen/antibody, hapten/antibody, lectin/carbohydrate,apoprotein/cofactor and biotin/(strept)avidin. The assays are oftenperformed with test strips in which a specific binding pair member isattached to a mobilizable material (such as a metal sol or beads made oflatex or glass) or an immobile substrate (such as glass fibers,cellulose strips or nitrocellulose membranes). Particular examples ofsome of these assays are shown in U.S. Pat. Nos. 4,703,017; 4,743,560;and 5,073,484 (incorporated herein by reference). The test stripsinclude a flow path from an upstream sample application area to a testsite. For example, the flow path can be from a sample application areathrough a mobilization zone to a capture zone. The mobilization zone maycontain a mobilizable marker that interacts with an analyte or analyteanalog, and the capture zone contains a reagent that binds the analyteor analyte analog to detect the presence of an analyte in the sample.

Examples of migration assay devices, which usually incorporate withinthem reagents that have been attached to colored labels, therebypermitting visible detection of the assay results without addition offurther substances are found, for example, in U.S. Pat. No. 4,770,853;WO 88/08534; and EP-A 0 299 428 (incorporated herein by reference).There are a number of commercially available lateral-flow type tests andpatents disclosing methods for the detection of large analytes (MWgreater than 1,000 Daltons) as the analyte flows through multiple zoneson a test strip. Examples are found in U.S. Pat. No. 5,229,073(measuring plasma lipoprotein levels), and U.S. Pat. Nos. 5,591,645;4,168,146; 4,366,241; 4,855,240; 4,861,711; 5,120,643; European PatentNo. 0296724; WO 97/06439; WO 98/36278; and WO 08/030546 (each of whichare herein incorporated by reference). Multiple zone lateral flow teststrips are disclosed in U.S. Pat. No. 5,451,504, U.S. Pat. No.5,451,507, and U.S. Pat. No. 5,798,273 (incorporated by referenceherein). U.S. Pat. No. 6,656,744 (incorporated by reference) discloses alateral flow test strip in which a label binds to an antibody through astreptavidin-biotin interaction.

In particular examples, the methods disclosed herein include applicationof a biological sample (such as serum, whole blood or urine) from ahuman test subject to a lateral flow test device for the detection ofone or more molecules (such as one or more molecules associated withmelioidosis, for example, combinations of molecules as described above)in the sample. The lateral flow test device includes one or moreantibodies (such as antibodies that bind one or more of the moleculesassociated with melioidosis) at an addressable location. In a particularexample, the lateral flow test device includes antibodies that bind atleast one melioidosis protein antigen listed in Table 1. The addressablelocations can be, for example, a linear array or other geometric patternthat provides diagnostic information to the user. The binding of one ormore molecules in the sample to the antibodies present in the testdevice is detected and the presence or amount of one or more moleculesin the sample of the test subject is compared to a control, wherein achange in the presence or amount of one or more molecules in the samplefrom the test subject as compared to the control indicates that thesubject has a B. pseudomallei associated condition, such as melioidosis.

Devices described herein generally include a strip of absorbent material(such as a microporous membrane), which, in some instances, can be madeof different substances each joined to the other in zones, which may beabutted and/or overlapped. In some examples, the absorbent strip can befixed on a supporting non-interactive material (such as nonwovenpolyester), for example, to provide increased rigidity to the strip.Zones within each strip may differentially contain the specific bindingpartner(s) and/or other reagents required for the detection and/orquantification of the particular analyte being tested for, for example,one or more molecules disclosed herein. Thus these zones can be viewedas functional sectors or functional regions within the test device.

In general, a fluid sample is introduced to the strip at the proximalend of the strip, for instance by dipping or spotting. A sample iscollected or obtained using methods well known to those skilled in theart. The sample containing the particular molecules to be detected maybe obtained from any biological source. Examples of biological sourcesinclude blood serum, blood plasma, urine, BALF, spinal fluid, saliva,fermentation fluid, lymph fluid, tissue culture fluid and ascites fluidof a human or animal. In a particular example, the biological source issaliva. In one particular example, the biological source is whole blood,such as a sample obtained from a finger prick. The sample may bediluted, purified, concentrated, filtered, dissolved, suspended orotherwise manipulated prior to assay to optimize the immunoassayresults. The fluid migrates distally through all the functional regionsof the strip. The final distribution of the fluid in the individualfunctional regions depends on the adsorptive capacity and the dimensionsof the materials used.

Another common feature to be considered in the use of assay devices is ameans to detect the formation of a complex between an analyte (such asone or more molecules described herein) and a capture reagent (such asone or more antibodies). A detector (also referred to as detectorreagent) serves this purpose. A detector may be integrated into an assaydevice (for example included in a conjugate pad, as described below), ormay be applied to the device from an external source.

A detector may be a single reagent or a series of reagents thatcollectively serve the detection purpose. In some instances, a detectorreagent is a labeled binding partner specific for the analyte (such as agold-conjugated antibody for a particular protein of interest, forexample those described herein).

In other instances, a detector reagent collectively includes anunlabeled first binding partner specific for the analyte and a labeledsecond binding partner specific for the first binding partner and soforth. Thus, the detector can be a labeled antibody specific for aprotein described herein. The detector can also be an unlabeled firstantibody specific for the protein of interest and a labeled secondantibody that specifically binds the unlabeled first antibody. In eachinstance, a detector reagent specifically detects bound analyte of ananalyte-capture reagent complex and, therefore, a detector reagentpreferably does not substantially bind to or react with the capturereagent or other components localized in the analyte capture area. Suchnon-specific binding or reaction of a detector may provide a falsepositive result. Optionally, a detector reagent can specificallyrecognize a positive control molecule (such as a non-specific human IgGfor a labeled Protein A detector, or a labeled Protein G detector, or alabeled anti-human Ab(Fc)) that is present in a secondary capture area.

Flow-Through Device Construction and Design

Representative flow-through assay devices are described in U.S. Pat.Nos. 4,246,339; 4,277,560; 4,632,901; 4,812,293; 4,920,046; and5,279,935; U.S. Patent Application Publication Nos. 20030049857 and20040241876; and WO 08/030546. A flow-through device involves a capturereagent (such as one or more antibodies) immobilized on a solid support,typically, a membrane (such as, nitrocellulose, nylon, or PVDF).Characteristics of useful membranes have been previously described;however, it is useful to note that in a flow-through assay capillaryrise is not a particularly important feature of a membrane as the samplemoves vertically through the membrane rather than across it as in alateral flow assay. In a simple representative format, the membrane of aflow-through device is placed in functional or physical contact with anabsorbent layer (see, e.g., description of “absorbent pad” below), whichacts as a reservoir to draw a fluid sample through the membrane.Optionally, following immobilization of a capture reagent, any remainingprotein-binding sites on the membrane can be blocked (either before orconcurrent with sample administration) to minimize nonspecificinteractions.

In operation of a flow-through device, a fluid sample (such as a bodilyfluid sample) is placed in contact with the membrane. Typically, aflow-through device also includes a sample application area (orreservoir) to receive and temporarily retain a fluid sample of a desiredvolume. The sample passes through the membrane matrix. In this process,an analyte in the sample (such as one or more protein, for example, oneor more molecules described herein) can specifically bind to theimmobilized capture reagent (such as one or more antibodies). Wheredetection of an analyte-capture reagent complex is desired, a detectorreagent (such as labeled antibodies that specifically bind one or moremolecules) can be added with the sample or a solution containing adetector reagent can be added subsequent to application of the sample.If an analyte is specifically bound by capture reagent, a visualrepresentative attributable to the particular detector reagent can beobserved on the surface of the membrane. Optional wash steps can beadded at any time in the process, for instance, following application ofthe sample, and/or following application of a detector reagent.

Lateral Flow Device Construction and Design

Lateral flow devices are commonly known in the art. Briefly, a lateralflow device is an analytical device having as its essence a test strip,through which flows a test sample fluid that is suspected of containingan analyte of interest. The test fluid and any suspended analyte canflow along the strip to a detection zone in which the analyte (ifpresent) interacts with a capture agent and a detection agent toindicate a presence, absence and/or quantity of the analyte.

Numerous lateral flow analytical devices have been disclosed, andinclude those shown in U.S. Pat. Nos. 4,168,146; 4,313,734; 4,366,241;4,435,504; 4,775,636; 4,703,017; 4,740,468; 4,806,311; 4,806,312;4,861,711; 4,855,240; 4,857,453; 4,861,711; 4,943,522; 4,945,042;4,496,654; 5,001,049; 5,075,078; 5,126,241; 5,120,643; 5,451,504;5,424,193; 5,712,172; 6,555,390; 6,258,548; 6,699,722; 6,368,876 and7,517,699; EP 0810436; EP 0296724; WO 92/12428; WO 94/01775; WO95/16207; WO 97/06439; WO 98/36278; and WO 08/030546, each of which isincorporated by reference. Further, there are a number of commerciallyavailable lateral flow type tests and patents disclosing methods for thedetection of large analytes (MW greater than 1,000 Daltons). U.S. Pat.No. 5,229,073 describes a semiquantitative competitive immunoassaylateral flow method for measuring plasma lipoprotein levels. This methodutilizes a plurality of capture zones or lines containing immobilizedantibodies to bind both the labeled and free lipoprotein to give asemi-quantitative result. In addition, U.S. Pat. No. 5,591,645 providesa chromatographic test strip with at least two portions. The firstportion includes a movable tracer and the second portion includes animmobilized binder capable of binding to the analyte.

Many lateral flow devices are one-step lateral flow assays in which abiological fluid is placed in a sample area on a bibulous strip (thoughnon-bibulous materials can be used, and rendered bibulous, e.g., byapplying a surfactant to the material), and allowed to migrate along thestrip until the liquid comes into contact with a specific bindingpartner (such as an antibody) that interacts with an analyte (such asone or more molecules) in the liquid. Once the analyte interacts withthe binding partner, a signal (such as a fluorescent or otherwisevisible dye) indicates that the interaction has occurred. Multiplediscrete binding partners (such as antibodies) can be placed on thestrip (for example in parallel lines) to detect multiple analytes (suchas two or more molecules) in the liquid. The test strips can alsoincorporate control indicators, which provide a signal that the test hasadequately been performed, even if a positive signal indicating thepresence (or absence) of an analyte is not seen on the strip.

The construction and design of lateral flow devices is very well knownin the art, as described, for example, in Millipore Corporation, A ShortGuide Developing Immunochromatographic Test Strips, 2nd Edition, pp.1-40, 1999, available by request at (800) 645-5476; and Schleicher &Schuell, Easy to Work with BioScience, Products and Protocols 2003, pp.73-98, 2003, 2003, available by request at Schleicher & SchuellBioScience, Inc., 10 Optical Avenue, Keene, N. H. 03431, (603) 352-3810;both of which are incorporated herein by reference.

Lateral flow devices have a wide variety of physical formats that areequally well known in the art. Any physical format that supports and/orhouses the basic components of a lateral flow device in the properfunction relationship is contemplated by this disclosure.

In some embodiments, the lateral flow strip is divided into a proximalsample application pad, an intermediate test result zone, and a distalabsorbent pad. The flow strip is interrupted by a conjugate pad thatcontains labeled conjugate (such as gold- or latex-conjugated antibodyspecific for the target analyte or an analyte analog). A flow path alongstrip passes from proximal pad, through conjugate pad, into test resultzone, for eventual collection in absorbent pad. Selective binding agentsare positioned on a proximal test line in the test result membrane. Acontrol line is provided in test result zone, slightly distal to thetest line. For example, in a competitive assay, the binding agent in thetest line specifically binds the target analyte, while the control lineless specifically binds the target analyte.

In operation of the particular embodiment of a lateral flow device, afluid sample containing an analyte of interest, such as one or moremolecules described herein (for example, protein antigens listed inTable 1, as discussed above), is applied to the sample pad. In someexamples, the sample may be applied to the sample pad by dipping the endof the device containing the sample pad into the sample (such as serumor urine) or by applying the sample directly onto the sample pad (forexample by placing the sample pad in the mouth of the subject). In otherexamples where a sample is whole blood, an optional developer fluid isadded to the blood sample to cause hemolysis of the red blood cells and,in some cases, to make an appropriate dilution of the whole bloodsample.

From the sample pad, the sample passes, for instance by capillaryaction, to the conjugate pad. In the conjugate pad, the analyte ofinterest, such as a protein of interest, may bind (or be bound by) amobilized or mobilizable detector reagent, such as an antibody (such asantibody that recognizes one or more of the molecules described herein).For example, a protein analyte may bind to a labeled (e.g.,gold-conjugated or colored latex particle-conjugated) antibody containedin the conjugate pad. The analyte complexed with the detector reagentmay subsequently flow to the test result zone where the complex mayfurther interact with an analyte-specific binding partner (such as anantibody that binds a particular protein, an anti-hapten antibody, orstreptavidin), which is immobilized at the proximal test line. In someexamples, a protein complexed with a detector reagent (such asgold-conjugated antibody) may further bind to unlabeled, oxidizedantibodies immobilized at the proximal test line. The formation of acomplex, which results from the accumulation of the label (e.g., gold orcolored latex) in the localized region of the proximal test line isdetected. The control line may contain an immobilized,detector-reagent-specific binding partner, which can bind the detectorreagent in the presence or absence of the analyte. Such binding at thecontrol line indicates proper performance of the test, even in theabsence of the analyte of interest. The test results may be visualizeddirectly, or may measured using a reader (such as a scanner). The readerdevice may detect color or fluorescence from the readout area (forexample, the test line and/or control line).

In another embodiment of a lateral flow device, there may be a second(or third, fourth, or more) test line located parallel or perpendicular(or in any other spatial relationship) to test line in test result zone.The operation of this particular embodiment is similar to that describedin the immediately preceding paragraph with the additionalconsiderations that (i) a second detector reagent specific for a secondanalyte, such as another antibody, may also be contained in theconjugate pad, and (ii) the second test line will contain a secondspecific binding partner having affinity for a second analyte, such as asecond protein in the sample. Similarly, if a third (or more) test lineis included, the test line will contain a third (or more) specificbinding partner having affinity for a third (or more) analyte.

1. Sample Pad

The sample pad is a component of a lateral flow device that initiallyreceives the sample, and may serve to remove particulates from thesample. Among the various materials that may be used to construct asample pad (such as glass fiber, woven fibers, screen, non-woven fibers,cellosic fibers or paper), a cellulose sample pad may be beneficial if alarge bed volume (e.g., 250 μl/cm²) is a factor in a particularapplication. Sample pads may be treated with one or more release agents,such as buffers, salts, proteins, detergents, and surfactants. Suchrelease agents may be useful, for example, to promote resolubilizationof conjugate-pad constituents, and to block non-specific binding sitesin other components of a lateral flow device, such as a nitrocellulosemembrane. Representative release agents include, for example, trehaloseor glucose (1%-5%), PVP or PVA (0.5%-2%), Tween 20 or Triton X-100(0.1%-1%), casein (1%-2%), SDS (0.02%-5%), and PEG (0.02%-5%).

2. Membrane and Application Solution:

The types of membranes useful in a lateral flow device (such asnitrocellulose (including pure nitrocellulose and modifiednitrocellulose), nitrocellulose direct cast on polyester support,polyvinylidene fluoride, or nylon), and considerations for applying acapture reagent to such membranes have been discussed previously.

In some embodiments, membranes comprising nitrocellulose are preferablyin the form of sheets or strips. The thickness of such sheets or stripsmay vary within wide limits, for example, from about 0.01 to 0.5 mm,from about 0.02 to 0.45 mm, from about 0.05 to 0.3 mm, from about 0.075to 0.25 mm, from about 0.1 to 0.2 mm, or from about 0.11 to 0.15 mm. Thepore size of such sheets or strips may similarly vary within widelimits, for example from about 0.025 to 15 microns, or more specificallyfrom about 0.1 to 3 microns; however, pore size is not intended to be alimiting factor in selection of the solid support. The flow rate of asolid support, where applicable, can also vary within wide limits, forexample from about 12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about22.5 to 62.5 sec/cm (i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm(i.e., 100 to 250 sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250sec/4 cm), or about 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). Inspecific embodiments of devices described herein, the flow rate is about62.5 sec/cm (i.e., 250 sec/4 cm). In other specific embodiments ofdevices described herein, the flow rate is about 37.5 sec/cm (i.e., 150sec/4 cm).

3. Conjugate Pad

The conjugate pad serves to, among other things, hold a detectorreagent. Suitable materials for the conjugate pad include glass fiber,polyester, paper, or surface modified polypropylene. In someembodiments, a detector reagent may be applied externally, for example,from a developer bottle, in which case a lateral flow device need notcontain a conjugate pad (see, for example, U.S. Pat. No. 4,740,468).

Detector reagent(s) contained in a conjugate pad is typically releasedinto solution upon application of the test sample. A conjugate pad maybe treated with various substances to influence release of the detectorreagent into solution. For example, the conjugate pad may be treatedwith PVA or PVP (0.5% to 2%) and/or Triton X-100 (0.5%). Other releaseagents include, without limitation, hydroxypropylmethyl cellulose, SDS,Brij and β-lactose. A mixture of two or more release agents may be usedin any given application. In a particular disclosed embodiment, thedetector reagent in conjugate pad is a gold-conjugated antibody.

4. Absorbent Pad

The use of an absorbent pad in a lateral flow device is optional. Theabsorbent pad acts to increase the total volume of sample that entersthe device. This increased volume can be useful, for example, to washaway unbound analyte from the membrane. Any of a variety of materials isuseful to prepare an absorbent pad, for example, cellulosic filters orpaper. In some device embodiments, an absorbent pad can be paper (i.e.,cellulosic fibers). One of skill in the art may select a paper absorbentpad on the basis of, for example, its thickness, compressibility,manufacturability, and uniformity of bed volume. The volume uptake of anabsorbent made may be adjusted by changing the dimensions (usually thelength) of an absorbent pad.

The disclosure is illustrated by the following non-limiting Example.

EXAMPLE In vivo Microbial Antigen Discovery of Antigens Method

This example provides an in vivo Microbial Antigen Discovery (InMAD) foridentification of B. pseudomallei antigens that are shed into bodyfluids during infection. Antigens discovered in this manner are targetsfor immunoassays for diagnosis of B. pseudomallei associated conditions.

FIG. 1 provides a schematic of InMAD. In the first step, mice, bodyfluids, e.g., serum and urine were collected from the infected humansubject when the subject showed signs of clinical disease. Termed InMADserum or urine, these samples contain the B. pseudomallei antigens thatare potential targets for diagnosis of B. pseudomallei-associatedconditions, such as melioidosis. InMAD serum was filter-sterilized toremove whole cells, but leave behind soluble antigens released duringinfection. The filtered samples were used to immunize naïve animalsubjects and their serum was collected to identify antigens recognizedby antibodies using one dimensional and two dimensional immunoblotsprepared from whole cell lysates. Mass spectroscopy was used to identifythose reactive antigens.

FIG. 2A is a digital image of a two-dimensional blot of Burkholderiapseudomallei lysate probed with InMAD immune serum illustrating thetotal proteins from the B. pseudomallei. FIG. 2B is digital image of atwo-dimensional Western blot of B. pseudomallei lysate probed with InMADimmune serum from mice immunized with human urine. Twelve spots(proteins) not found after immunization with control serum or urine fromuninfected animals are circled. Each spot is a candidate diagnostictarget. Each number corresponds to the protein number in Table 1 shownbelow.

TABLE 1 B. pseudomallei protein antigens reactive with InMAD immuneserum by 2-D Western blot. Protein ID Size (kDa) 1 Co-chaperonin GroES11 gi 107021935 2 50s Ribosomal Subunit 13 gi 53723863 3 HypotheticalProtein 14 gi 217419383 4 Co chaperonin GroES 11 gi 107021935 5 Phasin20 gi 167581317 6 Hypothetical Protein/OsmY 20/22 gi 53718900/76819679 7Glutathione S-transferase domain containing 23 protein gi 76808775 8/9Electron transfer flavoprotein subunit alpha 32 gi 53720108 10 FtsA/FtsZ 43/42 gi 53720630/53720631 11  GroEL/Phosphopyruvate hydratase55/45 gi 167912219/53719880 12  Transcription Elongation Factor NusA 55gi 53719533

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method of identifying a diagnostic indicator, comprising:selecting a condition or disease for which a diagnostic assay is desiredand is believed to be associated with one or more antigens; immunizing aveterinary subject which is not afflicted with the selected condition ordisease with a human biological sample obtained from a human subjecthaving the selected condition or disease to generate antibodies;collecting a biological sample comprising the generated antibodies fromthe immunized veterinary subject; and identifying one or more diagnosticindicators for the selected condition or disease, wherein an alterationin at least one antigen detected by the generated antibodies in thesample obtained from the immunized veterinary subject relative to thecontrol indicates that such antigen is a diagnostic indicator for thecondition or disease.
 2. The method of claim 1, wherein the biologicalsample is a serum sample.
 3. The method of claim 1, wherein thebiological sample is a whole blood sample.
 4. The method of claim 1,further comprising obtaining the human biological sample from the humansubject with the selected condition or disease.
 5. The method of claim1, wherein the human biological sample is serum or blood sample.
 6. Themethod of claim 1, wherein the human biological sample is a urinesample.
 7. The method of claim 1, wherein the immunized veterinarysubject biological sample is blood, serum or urine.
 8. The method ofclaim 1, wherein identifying one or more diagnostic indicators for theselected condition or disease comprises using one-dimensional ortwo-dimensional immunoblots.
 9. The method of claim 1, whereinidentifying one or more diagnostic indicators for the selected conditionor disease comprises using one-dimensional or two-dimensionalimmunoblots followed by mass spectroscopy.